Process of increasing plant growth and yield and modifying cellulose production in plants

ABSTRACT

A process of increasing plant growth and yield comprises introducing into the plant a DNA sequence encoding a product which modifies, in the plant, the level of cellulose precursors.

FIELD OF THE INVENTION

[0001] The present invention relates to processes of enhancing plantgrowth and productivity and more specifically, to the field of carbonre-allocation in plants.

BACKGROUND OF THE INVENTION

[0002] Increasing harvestable plant yield is a major goal of all plantbreeding efforts. In fiber producing crops, the economic value of thisyield is directly related to the amount, location, and length of thecellulose fibers. It has been suggested that cellulose content and fiberyield is limited by the amount of substrate, or sugars, produced duringphotosynthesis. However, numerous studies provide evidence that althoughcrucial for plant growth and survival, the availability of carbohydratesderived from photosynthesis are not major limiting factors in cellulosesynthesis. Thus there exists a substantial opportunity to increase fiberyield by creating a sink for this existing photosynthate in cells highin cellulose. Sucrose, the major form of translocatable carbohydrateproduced during photosynthesis in the plant, is translocated to sinktissue where it is converted to other compounds such as starch orcellulose.

[0003] Despite the fact that the amount of photosynthates in the plantare not a primary limitation in cellulose content, the rate ofphotosynthesis plays a large role in the overall growth of a plant.Further, one element in the control of photosynthesis in the plant isthe feedback-inhibition of photosynthesis by photosynthetic products,such as starch, sucrose and hexose sugars. Goldschmidt and Huber (1992)¹tested the effect of girdling the leaves of crop plants and demonstratedthat the build up of starch and other products of photosynthesisactually inhibited the rate of photosynthesis. These findings, andothers (Sonnewald & Willmitzer 1992)², indicate the photosynthetic rate,and ultimately plant growth, may be directly correlated with the ratethat photosynthates are drawn away from the leaf, or the rate ofbiosynthetic degradation in the leaves. The degradation ofphotosynthates occurs primarily in cells/tissues that are activelygrowing (meristematic or young tissues) or in tissues wherephotosynthates are utilized for storage or structural components (sinktissues). Therefore altering the rate that carbohydrates aretranslocated to these sink tissues (altering carbon allocation) wouldnot only increase overall plant growth (remove inhibitors ofphotosynthesis), but also increase the amount of storage (starch) orstructural components (cellulose).

[0004] A striking example of the benefits of altering carbon allocationhas been demonstrated in potato. By increasing the synthesis andaccumulation of ADP-glucose in the tuber, starch synthesis increasedwhich significantly increased dry matter content. In fact, this resultedin a 25% increase in tuber yield. The increase in ADP-glucose in thetuber was accomplished by genetically engineering the potato with abacterial ADP-glucose pyrophosphorylase gene controlled by a tuberspecific promoter (Shewmaker and Stalker 1992)³.

[0005] Much like ADP-glucose is a precursor to starch synthesis, thenucleotide sugar UDP-Glucose, (UDPG), is a high energy substrate forcellulose biosynthesis in both bacteria and higher plants (Delmer 1987⁴,Delmer et al. 1995⁵). Several bacterial genes which encode the enzymeUDP-glucose pyrophosphorylase (UDPG-PPase), responsible for thesynthesis of UDPG, have been isolated (Ross et al. 1991⁶). An existingpatent by Betlach (1987⁷) claims increased synthesis of xanthan andother polysaccharides in bacteria by insertion of a UDPG-PPase gene fromXanthamonas campestris. However, the claims in this patent are limitedto increasing polysaccharide biosynthesis in prokaryotic organisms.

[0006] It is an object of the present invention to obviate or mitigatethe above disadvantages.

SUMMARY OF THE INVENTION

[0007] The present invention provides a process of increasing plantgrowth and yield which comprises introducing into a plant a DNA sequenceencoding a product which modifies, in the plant, the level of celluloseprecursors. This product of the present invention includes, but is notlimited to, ribonucleic acid (“RNA”) molecules, enzymes related tocellulose biosynthesis and proteins which regulate the expression ofthese enzymes.

[0008] It has been found that the process of the present invention leadsto the reallocation by simple diffusion of carbohydrates such as glucosefrom photosynthetic cells, such as the leaf cells, to other cells withinthe plant. This translocation removes the inhibition on photosynthesisimposed by excess photosynthate accumulation in these photosyntheticcells thereby allowing the plant to produce more simple sugars bycontinued photosynthesis. In other words, as photosynthesis continues inan uninhibited fashion, more simple sugars are produced than would haveotherwise have been possible. These simple sugars are building blocksfor plant growth via the production of polymers such as starch andcellulose.

[0009] Further, the present invention provides a process of modifyingthe production of cellulose in a plant which comprises introducing intosaid plant a DNA sequence encoding a product which modifies, in theplant, the level of cellulose substrates. As above, the productincludes, but is not limited to, ribonucleic acid (“RNA”) molecules,enzymes related to cellulose biosynthesis and proteins which regulatethe expression of these enzymes.

[0010] The subject invention also provides a plant having increasedgrowth and yield and/or modified cellulose producing activity as aresult of introducing into said plant or parent of said plant a DNAsequence coding for a product which modifies the level of celluloseprecursors in the plant.

[0011] Another aspect of the present invention provides for a DNAexpression vector comprising a DNA sequence encoding a product whichmodifies, in a host, the level of cellulose precursors, said sequencebeing operably linked to an expression effecting DNA sequence andflanked by translational start and stop sequences.

[0012] The present invention also provides a genetically modified seedcomprising a DNA sequence, said sequence encoding a product capable ofincreasing growth and yield and/or modifying the level of celluloseprecursors in the plant resulting from said seed.

[0013] There are two primary features of the process of the presentinvention. Firstly, in all plants regardless of whether they arefiber-producing (trees, hemp, cotton etc.) or not, what is achieved areplants having faster rates of growth and increased yield bynon-specifically re-allocating carbon within the plant away fromphotosynthetic cells. This allows photosynthesis to continue uninhibitedto produce more simple “construction” sugars thereby enhancing theefficiency of the plant growth rate and increasing growth yield.Secondly, in fiber-producing plants, the expression of the DNA sequenceintroduced into the plant may be targeted to specific individual celltypes within the plant to increase predictably cellulose deposition in acell specific manner. In forest trees, this is expected to increase woodproduction and fiber yield, especially when the gene is linked to apromoter which expresses only in wood forming tissues. Increased fiberyield can also be expected in other non-forestry fiber producing plants,such as hemp and sisal. In addition to targeting wood forming tissues,increased cellulose production can be obtained in other parts of theplant such as the boles surrounding the seeds of cotton plants.

[0014] Specific applications for increased cellulose synthesis includenumerous crops with diverse uses and growth habits. In forestry, woodproduction is influenced by a combination of physiological andbiochemical processes governed by substantial genetic variation. Thishas lead to the theoretical consideration of limitations on increasingyield due to fundamental constraints on energy supply (Farnum 1983⁸).Despite such limitations, increases in tree growth of 50 to 300% arepossible depending on the tree species and growing environment. Clearly,improving energy capture, conversion of radiant energy, and alteringcarbon allocation within the plant are promising areas for treeimprovement. Increasing cellulose content by the processes outlinedherein can achieve such gains.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is described by way of the followingnon-limiting drawings in which:

[0016]FIG. 1 illustrates schematically the formation gene cassettecomprising the UDPG-PPase gene and CaMV promoter, the cloning vector pUCcomprising the gene cassette and transformation vectors pBI121 and pAX6comprising the gene cassette and preferential promoters;

[0017]FIG. 2 represents the nucleotide sequence of the cloned genecassettes in the pBI series of binary vectors;

[0018]FIG. 3 illustrates schematically the formation of the xylemspecific transformation vector with the UDPG-PPase gene;

[0019]FIG. 4 represents an assay of UDPG-PPase activity in tobaccoplants;

[0020]FIG. 5 is a graph representing the titre of anti-UDPG-PPase serawith affinity purification;

[0021]FIG. 6 is a Western Blot analysis of UDPG-PPase protein with theanti-UDPG-PPase antibody; and

[0022]FIG. 7 is a bar graph representing an analysis of cellulose intransformed tobacco plants.

PREFERRED EMBODIMENTS OF THE INVENTION

[0023] The present invention affords the ability to increase plantgrowth rates and yield through the addition to the plants of DNAsequences encoding products which have a modifying role on the level ofcellulose precursors. The result of the introduction and expression inthe plant of this DNA sequence is the beneficial and optionallyselective allocation of carbon within the plant.

[0024] In a preferred form of the invention described furtherhereinbelow, the DNA sequence may be selectively expressed in cellsprimarily responsible for cellulose synthesis. By creating a sink forthese carbohydrates in cellulose producing cells, excess photosynthatecan be diverted to these cells where the default pathway for their usewould be conversion into cellulose, thereby increasing cellulose contentin the plant. For example, in trees, as photosynthate such as sucroseand hexose sugars are removed from the leaves to the stem the inhibitoryeffect of these compounds on photosynthesis is removed. This has hugeimplications in forestry, because whether harvesting for lumber orfiber, the product is cellulose. The benefit of increased cellulose isnot limited, however, to forestry, as there are numerous other fibercrops including sisal, cotton, and hemp.

[0025] In one embodiment of the present invention, the product encodedby the inserted DNA sequence is an enzyme such as acarbohydrate-modifying enzyme selected from the group consisting ofuridine diphosphate-glucose pyrophosphorylase (“UDPG-PPase”), sucrosesynthetase, cellulose synthase or any derivative thereof. Sucrosesynthetase is responsible for the synthesis of uridinediphosphate-glucose (“UDP-glucose”) in plants. The present applicationis not limited to the specific enzymes disclosed herein as these areintended merely as a sampling of preferred enzymes. What is required forthe enzymes to be useful herein is that they have the potential toeffect, in some way, the level of cellulose precursors in the plant.These enzymes may originate from any organism including other plantspecies, bacteria or yeast.

[0026] Although bacterial enzymes are preferred for the reasonsdescribed below, it is to be understood that the DNA sequences encodingenzymes may originate from many other organisms. The key criteria inselecting a “preferred” enzyme is a relatively high Km value for theproduct as compared to the precursors or substrates thereby indicating apreference in the reaction toward the product.

[0027] UDP-PPase is the most preferred enzyme particularly when the DNAsequence encoding the enzyme originates from bacteria. The enzymekinetics data (UDPG-PPase has a relatively high Km value for UDPG ascompared to the substrates UTP, glucose-1-phosphate and PPi), lack ofsignal sequences and the fact that, unlike the corresponding plant gene,the bacterial UDPG-PPase gene is not strongly inhibited by UDPGaccumulation make the bacterial UDPG-PPase gene an excellent target geneto increase UDPG levels in plants. Additionally, bacterial genes arewidely available and are less likely to lead to co-suppression of thenative UDPG-PPase genes. Bacterial genes may be selected from manycommonly available genera, but in a preferred form are selected from thegenus Acetobacter, more specifically from the species includingAcetobacter xylinum and from the genus Xanthomonas.

[0028] In an alternative embodiment, the DNA sequence introduced intothe plant may encode regulatory, feedback or other proteins which effectcellulose biosynthesis in plants or any derivatives thereof. Theseinclude lignin-modifying proteins and proteins which regulatelignin-modifying proteins.

[0029] In a further embodiment, the DNA sequence introduced into theplant encodes for an RNA molecule having regulatory properties. Forexample, these RNA molecules may effect enzyme synthesis, cellulosesynthesis or may indirectly modify cellulose synthesis through analteration in precursor or lignin synthesis.

[0030] Prior to the introduction of the DNA sequence into the plantcells as described further hereinbelow, the DNA sequence or gene ofinterest (terms “DNA sequence” and “gene” used hereinafterinterchangeably) encoding for the product with cellulose modulatoryeffects is prepared into a DNA construct or vector. Initially, the geneof interest is extracted by known techniques from the source (forexample, bacteria, yeast or other plant species) or obtained from adepository such as ATCC. The general extraction procedure involveslysing the cells of the source and recovering the released DNA throughextraction such as phenol/chloroform with a final precipitation in, forexample, alcohol.

[0031] The gene or DNA sequence is then amplified by, for example, thepolymerase chain reaction (“PCR”) and subsequently cloned into thedesired construct or vector. The amplification of the gene based on thePCR makes use of primers and inducing agents, sometimes referred to asenzyme catalysts. The PCR process is described in considerable detail inU.S. Pat. No. 4,800,159 and Canadian Patent No. 1,237,685 both to CetusCorporation and in U.S. Pat. Nos. 4,965,188 and 4,682,202 all of whichare incorporated herein by reference.

[0032] The term “primer” as used herein refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, which is capable of acting as a point ofinitiation of DNA synthesis when placed under conditions in whichsynthesis of a primer extension product which is complementary to thenucleotide sequence is induced, i.e. in the presence of nucleotides andinducing agent and at a suitable pH and temperature. The primer ispreferable single-stranded for maximum efficiency in amplication but mayalternatively be double-stranded. If double-stranded, the primer isfirst treated to separate its strands before being used to prepare theextention products. Preferably, the primer is anoligodeoxyribonucleotide. The exact lengths of the primers may bedifferent for each DNA sequence or “template” to be amplified.Generally, a balance must be struck with respect to primer size. It mustbe large enough to be usefully specific to the template, that is, itmust be homologous to a large enough region of the template so thatother extraneous DNA (not related to the DNA sequence) with some degreeof homology to the primer is not amplified to a significant extent. Onthe other hand, the size of the primer should not be so large as to beunwieldy and prohibitive in terms of time and cost. This balance may beachieved for most of the DNA sequences contemplated within the scope ofthe present invention with primer of between 10-50 nucleotides inlength. The determination of the appropriate lengths of primers,however, is well within the purview of a technician of average skill inthis area. In addition, although the PCR is an efficient process forproducing exponential quantities of a DNA product relative to the numberof reaction steps involved, other known DNA amplification techniques maybe used within the scope of the present invention.

[0033] Suitable constructs or vectors for transforming the plant hostare well known in the art and include plasmids, cosmids, phagederivatives, phasmids and expression vectors. General vectors ofinterest may contain an origin of replication functional in one or moreplant species, convenient restriction endonuclease digestion sites andselectable markers for the plant cell. Preferred transformation vectorsvary depending on the particular host but include Bluescript vectors,pBI (Agrobacterium binary vectors) and pUC derived vectors. Othervectors useful for assessing MRNA and protein expression in plantsinclude pMAL and pGEM vectors.

[0034] In order to achieve expression of the DNA sequence of interest ina plant host, it may be necessary to make modifications to theregulatory and/or controlling sequences of that DNA. Specifically, itmay be necessary to link the gene of interest operably to an expressioneffecting DNA sequence, such as one or more promoters and to flank itwith translational start and stop signals. In particular, the startcodon may be changed and suitable plant promoter and terminatorsequences added. Optionally, an improved translation consensus sequencesmay be provided. It is to be understood, however, that thesemodifications need not be made for each and every DNA sequencecontemplated within the scope of the present invention. The question ofmaking these technical modifications is well within the purview of atechnician of average skill in this field.

[0035] A number of promoters may be ligated to the DNA sequence, themost efficient type of which varies between plant hosts. In a preferredform, the promoter expresses specifically in vascular plant cells orcellulose-producing cells within the plant. For example, in trees, xylemspecific promoters including, but not limited to the 4-coumarate CoAligase (“4CL”) promoter from parsley are preferred in order to directexpression to wood-forming tissues. In tobacco plants, suitablepromoters include the cauliflower mosaic virus (“CaMV”) 35S promoter. Inother plant species, 4CL and CaMv 35S among others may be used.

[0036] For consistency in terminology, the DNA sequence to betransformed having modifications to the regulatory and/or controllingsequences is referred to hereinafter as a “gene cassette” or “DNAsequence cassette”. Transformation of this DNA sequence cassette into aplant host may be achieved by a number of established methods. Generallyfor most plants including tobacco, the widely practised Agrobacteriumtransformation method is appropriate. General techniques fortransformation of plants can be found in Svab Z. P. Hajdukiewicz and P.Maliga. 1995. Generation of Transgenic Tobacco Plants by AgrobacteriumTransformation. pp. 61-77. (eds. P. Maliga, D. F. Klessig, A. R.Cashmore, W. Gruissem and J. E. Varner) Methods in Plant MolecularBiology, Cold Spring Harbor Laboratory Press, New York and Horsch, R.B., J. E. Fry, N. L. Hoffman, D. Eichholtz, S. G. Rogers and R. T.Fraley. 1985. A Simple and General Method for Transferring Genes intoPlants. Science 227:1229-1231 both of which are incorporated herein byreference. In a preferred form for trees, in particular coniferousspecies such as spruce, the particle gun bombardment method may be usedin conjunction with embryonic cultures.

[0037] In the particle gun bombardment process, which is described inmore detail in the incorporated reference: Ellis et al. 1993. StableTransformation of Picea glauca by Particle Acceleration. Bio/Technology.vol. 11 pp. 84-89, embryonic cultures of the plant host are exposed, forshort time, to a blast or bombardment of the DNA sequence or DNAsequence cassette to be transformed. Generally, this is achieved byinert gas (such as helium) propulsion of micro-particles of gold coatedwith the DNA sequence to be transformed. Optionally, the DNA sequencemay be fused to a marker gene, such as an antibiotic resistance gene toallow for subsequent selection of cultures for further regeneration. Forexample, the DNA sequence may be fused to a kanamycin resistance geneand the transformed cultures thereafter selected for plant regenerationon the basis of kanamycin resistance.

[0038] After transformation, the plant tissue is preferably placed on anantibiotic containing medium on which the transformed cells expressing aresistance gene are able to grow. Non-transformed cells are therebyretarded in their growth and/or die on the antibiotic. In this manner,once plants are regenerated either through the formation of shoots orthe development of mature embryos and germination (as is the case withsomatic embryogenesis), only plants capable of expressing the introducedgenes (the DNA sequence of the present invention together with theantibiotic resistance gene) are produced. The seeds (includingartificial seeds derived from somatic embryos) and subsequent plantsresulting from the transformation and regeneration process as describedherein have increased rates of growth and increased yields as a resultof the transformed DNA sequence which modifies the level of celluloseprecursors in the plant. For example, if the transformed DNA sequencecomprises the UDPG-PPase gene, glucose (a photosynthate) is converted inplant cells to UDP-glucose (a high energy substrate for celluosebiosynthesis). As these plant cells then have a lesser concentration ofglucose relative to photosynthetic cells, glucose translocates by simplediffusion to these cells. This carbon translocation reduces theinhibition of excess photosynthate on photosynthesis leading to moreefficient photosynthesis and enhanced sugar production. In a preferredform, when the DNA sequence expression is targeted to vascular orcellulose-producing cells via specific promoters, not only is therecarbon re-allocation as described above, but there is provided moreUDP-glucose in these cells allowing for enhance cellulose productionwith the attendant advantages.

EXAMPLES

[0039] Summary

[0040] Transformation of tobacco with a construct for overexpression ofan Acetobacter xylinum (Ax) UDPG-PPase gene has resulted in increaseddry weight, solute content, and a-cellulose content in the transformantsrelative to non-transformed control plants. Antibody specific to the AxUDPG-PPase gene has been generated in rabbits following injection of afusion protein overproduced in E. coli. Using this antibody, detectionof expressed UDPG-PPase in transgenic tobacco has been confirmed. Theinserted gene cassette segregated based on kanamycin resistance in mostof the T₁, population in a manner consistent with a single insertionsite. Initial experiments with the transformation of two constructs, a4CL-GUS and a 4CL-UDPG-PPase in spruce has yielded numerous putativetransformed lines. These lines are currently undergoing GUS screening(4CL-GUS) and further kanamycin screening (both constructs).

Example 1 Preparation of DNA Constructs

[0041] The original bacterium (Acetobacter xyliniim) containing the geneUDP-glucose pyrophosphorylase (UDPG-PPase) was obtained from ATCC(23768). The UDPG-PPase gene was amplified by PCR and subsequentlycloned. Design of the PCR primers included the following considerations:

[0042] Addition of restriction sites suitable for cloning into a varietyof transformation vectors.

[0043] Mutation of the start codon from valine to methionine.

[0044] Mutation of internal Eco RI site to remove it without change inamino acid sequence.

[0045] Addition of non-coding DNA fragment at the 5′ end of the gene toenhance the efficiency of translation.

[0046] The resulting primer A at the 5′ end is:

[0047] M A K P L K K A V L P

[0048] taGGATCCgtcgaccATGGTCAAccccttaaaaaagccgtattgc

[0049] and the original UDPG-PPase gene at 5′ end is:

[0050] ttgaggtaaatattaGTGATTAAgccccttaaaaaagccgtattgccggttg-->

[0051] V I K P L K K A V L P

[0052] The original UDPG-PPase gene at the 3′ end is:

[0053] ggtgccggaagatcacttgtacttcgtcaggaattcacgcacgccggg

[0054] Stop code * S N V C A P

[0055] and the primer B at the 3′ end is:

[0056] ggtgccTCTAGAtcACTTGTacttcgtcagGACTTCacgcacgccggg

[0057] Stop code * S N* V C A P

[0058] Amplification of the gene was successful and DNA sequencingconfirmed that the amplified fragment was the UDPG-PPase gene. Theamplified UDPG-PPase gene from A. xylinum is almost identical to thepublished sequences.

[0059] The complete DNA sequence has been analyzed to detect potentialexon and intron splice sites. Several characters have been considered tofind introns which could potentially cause splicing of the mRNA. Theseinclude no-random codons in the DNA sequence, preferential usage ofcertain codons, GC content, relative positions of purines andpyrimidines in the codons of known sequences, and exon value of regionsbetween splice sites, as well as downstream and upstream of the end ofthe exon. Analysis showed that the potential of mRNA splicing in planttissue was low.

[0060] The amplified UDPG-PPase sequence was cloned into a BlueScriptvector and used for construction of the gene cassettes. The genecassette was constructed by ligating the UDPG-PPase gene with a CaMV 35Spromoter. A pUC based cloning vector containing the UDPG-PPase gene withsuitable restriction sites for placing the “gene cassette” in a varietyof transformation vectors containing xylem preferential and otherpromoters was made. FIG. 1 shows that gene cassettes and vectors.

[0061] Several vectors containing the UDPG-PPase gene from Acetobacterxylinum (Ax) have been derived from the gene cassette for differentpurposes, as follows:

[0062] For Tobacco Transformation

[0063] pBIAx—Agrobacterium binary vector with the Ax gene linked to CaMV35S promoter

[0064] pBI4CLAx—Agrobacterium binary vector with the Ax gene linked to aparsley 4CL promoter for xylem preferential expression

[0065] For Spruce Transformation

[0066] pBI4CLAX—pUC-derived vector, containing Ax linked to 4CL promoter

[0067] p4CLGUS—pUC-derived vector containing GUS linked to a parsley 4CLpromoter for assessment of xylem specificity of the promoter

[0068] For Assessing mRNA and Protein Expression in Transformants

[0069] pMAL—protein expression vector in E. coli, for antibodyproduction.

[0070] PGem—MRNA transcriptional vector, for in situ hybridization.

[0071] pBI based binary vectors have been constructed by ligation ofUDPG-PPase gene into pBI121 for Agrobacterium transformation. Theresultant binary vector contains a transcriptional fusion of theUDPG-PPase gene to the CaMV 35S promoter. The identity of the clonedgene cassettes in the pBI series of binary vector was confirmed by DNAsequencing (FIG. 2). The 4-coumarate CoA ligase (4CL) promoter fromparsley has been identified as a xylem preferential promoter and the 4CLpromoter is highly specific for xylem expression in transgenic tobacco.The 4CL promoter was modified and ligated to the UDPG-PPase gene. Theconstruct was subsequently placed in a binary vector containing the 4CLpromoter fused to the UDPG-PPase gene. The details of vector map isshown in FIG. 3.

[0072]E. coli Expression Vector

[0073] An expression vector was constructed to raise antibodies forsubsequent analysis of transformed plants. The expression vector wasbased on the fusion of the UDPG-PPase reading frame to a maltose bindingdomain (MBP). This pMAL vector provides a method for expression andpurifying the UDPG-PPase protein in E. coli and is a commerciallyavailable vector allowing subsequent purification using a maltose columnfollowed by cleavage to obtain the original UDPG-PPase protein.

Example 2 Tobacco Transformation and Characterization of UDPG-PPaseExpression

[0074] Transformation

[0075] The binary vector, pBIAX6 containing the Ax UDPG-PPase gene, wastransformed into A. tumefaciens strain EHA 105 and this was used toinfect Nicotiana tabacum c.v. xanthii leaf discs. More than 42independent T₀ transformants were regenerated and individual plants weretransferred from tissue culture into a growth room for production ofseed. The stable transformation of the UDPG-PPase gene in the T₀ tobaccoplants was first confirmed by PCR amplification with internal primers(see previous report). Further analyses were carried out by SouthernBlot analysis. More than 42 independent transformed plants (T₀) haverooted and grown in both sterile MS medium and in soil. Seeds from 24 T₀plants have been harvested and used to generate T₁ plants. T₁ plantswere grown in soil following germination on kanamycin (150 mg/ml).Segregation of kanamycin resistant T₁ plants followed expectedsegregation patterns. The results demonstrated that the UDPG-PPase genewas successfully integrated into the tobacco genome. Activity of theexpressed UDPG-PPase gene was assayed in vivo and in vitro bymeasurement of NADPH formation accompanying the enzyme-coupledconversion to 6-phosphogluconate through G-6-P. In order to test theactivity of UDPG-PPase, tobacco leaves from the greenhouse were sampledusing a cork borer and ground to a powder with PVPP/sand in liquidnitrogen. The enzyme was extracted with magnesium/glycine-glycine bufferand added into the assay buffer. The formation of NADPH was monitored at340 nm at 30° C. continuously until a loss of the initial linearreaction rate occurred. The enzyme assay showed that UDPG-PPase activitywas significantly higher in transgenic tobacco carrying the UDPG-PPasegene compared to control plants (FIG. 4). Note that in FIG. 4 activityrefers to specific activity (units/mg protein). Standard refers to purecommercial enzyme preparation. TABLE 1 Summary of height growth (cm) oftobacco plants transformed with UDPG-PPase gene versus controls. Allplant were regenerated from leaf discs. Control Transformed Days of n =9 n = 42 % of growth Avg. SE Avg. SE control  1  9.02 0.94  9.67 0.40107% 20 12.25 1.16 12.54 0.43 102%  34^(a) 19.60 1.55 22.61 0.54 115% 4728.60 2.06 29.17 0.80 102%

[0076] Preliminary data on the height growth of the Tplants over a sixweek period is contained in Table 1. During the exponential growth phasethe transformed plants were significantly taller than the controls(P=0.05).

[0077] Segregation Analysis of T₁ Generation

[0078] Seeds from 16 T₀ plants were harvested and used for generatingthe T₁ generation. The germination rate of the T₀ seeds ranged from52-93%, averaging 73%. Seeds for the T₁ plants were germinated on mediumcontaining 100 or 150 ug/ml kanamycin and segregation of kanamycinresistant seedlings was scored. A Pearson chi-square test showed thatmost of the transformed tobacco plants contained the inserted genes in asingle locus (and presumably in a single copy) due to a segregationratio of approximately 3 to 1 (Table 2). Scoring of kanamycin resistancein germinating T₁ seedlings was not straight forward. On water-agar, thegermination frequency was very low, further, nontransformed controls andtransformed seeds had similar germination frequencies. Conversely withthe use of ½ strength MS medium, germination in the presence ofkanamycin was high with all seeds, including the controls. Severalparameters including root growth, cotyledon color, seedling vigor,seedling size, and the presence of primary leaves were assessed.Currently the only reproducible and reliable method for determination ofkanamycin resistance in the seedlings is germination for three weeks on½ MS containing 150 ug/ml kanamycin and scoring resistance based on thepresence or absence of primary leaves. TABLE 2 Segregation of kanamycinresistant T₁ tobacco seedlings based on presence (tolerant) or absence(susceptible) of primary leaves. Seeds of T₀ lines (ug/mg kanamycin) Kanresistant Kan sensitive Kan + /K 6.01 (150) 39 13 3.0 6.03 (100) 35 142.5 6.05 (100) 35 12 2.8 6.06 (150) 26 15 1.7 6.07 (100) 35 12 2.9 6.08(150) 29 10 2.9 6.09 (150) 38 12 3.2 6.12 (150) 35 14 2.5 6.13 (150) 3610 3.6 6 14 (150) 34 12 2.8 6.15 (150) 29 8 3.6 6.23 (150) 30 14 2.16.24 (150) 30 18 1.7 6.25 (150) 34 15 2.3 6.33 (150) 33 11 3.0 6.42(150) 38 12 3.2 Control5 0 44 0.0 Control8 2 46 0.0

Example 3 Analysis of Protein Expression

[0079] Protein Production and Antigen Purification

[0080] The protein expression vector pMALAX was used to overproduce theUDPG-PPase-maltose binding protein (MBP) fusion in E. coli afterinduction with isopropylthiogalactoside (IPTG). A crude protein extractwas obtained with guanidine and urea buffers. Purification of theUDPG-PPase-MBP fusion protein was done by affinity chromatography usingan amylose resin affinity column, with elution of the purified fusionprotein from the column with 10 mM maltose. This purified fusion proteinwas confirmed to be a pure fraction based on SDS-PAGE and was used forantibody production in rabbits.

[0081] Antibody Production

[0082] The anti-UDPG-PPase antibody was produced by Enz-ProbeBiotechnology, Burnaby, B.C. after immunization of rabbits with thepurified UDPG-PPase-MBP fusion protein. A UDPG-PPase specific antibodywas prepared from the immunized rabbit serum by affinity purification inthe presence of excess maltose binding protein (to displace antibodieswhich react to this portion of the fused protein). The purified antibodywas used to detect the expression of proteins in Western Blottingexperiments.

[0083] Protein Analysis in Transgenic Tobacco

[0084] The protein hybridization was carried out according to Sambrooket al. (1989)⁹ with the affinity purified antibody used at a 1:500dilution. Antibody raised against the purified protein cross-reacted onwestern blots with the extracted UDPG-PPase protein from both bacteriaand transformed plants. Western blots showed that the antibody bound topeptides of 30 KDa and 90 kDa, corresponding to the UDPG-PPase peptidewith and without the MBP fusion protein respectively. Removal of the MBPportion of the fusion protein was done by digestion with factor Xa in amodified incubation buffer. Expression of the UDPG-PPase gene intransgenic tobacco plants was inferred by the recognition of theanti-UDPG-PPase antibody to a 30 KDa peptide in the transgenic plants.Antibody binding to a peptide of similar molecular mass has never beendetected in non-transformed plants.

[0085] Although not evident in FIG. 6 (the Western Blot of UDPG-PPaseprotein with the anti-UDPG-PPase antibody), there is cross-reaction ofthe anti-UDPG-PPase antibody with several other bands in the proteinprofile of tobacco. Despite numerous experiments to further purify theantibody and increase the specificity of this antibody, the backgroundstill persists. In fact, definitive detection of the UDPG-PPase proteinfrom individual transformed plants has been difficult because of thisbackground. However, cleavage of the fusion protein with factor Xa (asmentioned above) provides a method to obtain higher affinity antibody toUDPG-PPase protein.

Example 4 Cellulose Analysis of Transgenic Tobacco

[0086] Cellulose is one of the most important polysaccharides in tobaccoand its production is directly linked to UDPG-PPase. Cellulose analysisof T₀ plants were done with both whole plants and stems from matureflowering plants. Approximately 20 g (f.w.) of plant tissue wasextracted with azeotropic ethanol-benzene (1:2 w/w) in a Soxhletapparatus. After extraction, the solution was dried for soluble materialanalysis. The plant tissue was then ground and thoroughly mixed to makea homogenous sample. One gram of this sample was delignified with sodiumchlorite in weak acetic acid and the lignin was washed away by gradualfiltration. The entire polysaccharide fraction of the sample was used todetermine holocellulose. Removal of hemicellulose was performed bytreatment with 24% potassium hydroxide and the pure form ofalpha-cellulose was recovered as a white product from filtration througha sintered crucible.

[0087] Total biomass and cellulose analysis of five control and five T₀transformed (treated) plants showed that the transgenic plantscontaining the UDPG-PPase gene had significantly higher dry weight,solute content, and most importantly a-cellulose content (FIG. 7). Nosignificant differences in holocellulose were detected.

Example 5 Protein Expression and Antibody Production

[0088] The anti-UDPG-PPase rabbit serum was collected from rabbits afterimmunization with the UDPG-PPase protein produced in E. coli. The totalantibody was assayed with enzyme-labeled protein. Antibody activityagainst the purified protein was detected in serum by ELISA at a titreof 32,000; a working ELISA dilution of {fraction (1/500)} was used. AUDPG-PPase specific antibody, was then prepared by affinity purificationin the presence of excess maltose binding domain protein to displaceantibodies which react the MBP portion of the fused protein. The titreof anti-UDPG-PPase sera with affinity purification is shown in FIG. 5.Note that Fractions 2 & 3 obtained by elution from an affinity column.The titre dilution is {fraction (1/1)}, {fraction (1/500)}, {fraction(1/2000)}, {fraction (1/8000)}, {fraction (1/32000)}, {fraction(1/128000)}, {fraction (1/512000)}, {fraction (1/2048000)} contrastedwith pre-immune serum (titre series 2 to 9 respectively.) The purifiedantibody has identified a band on a Western Blot of the same molecularweight as the UDPG-PPase protein.

Example 6 Stable Transformation of Spruce With 4CL-UDPG

[0089] Transformation of UDPG-PPase Gene Into Spruce

[0090] Using biolistics, transformation of spruce somatic embryos withboth pBI4CLAX and pUC4CLGUS has been initiated. Over 1,500 interiorspruce and 200 Sitka spruce somatic embryos have been bombarded withthese constructs. The interior spruce embryos are from four differentgenotypes, and several different developmental stages. Followingparticle bombardment, the embryos were allowed to recover two weeksprior to placement on selective medium containing 5 mg/ml kanamycin.Embryos were transferred every three weeks onto fresh kanamycin mediumfor three transfers and then placed on kanamycin-free medium for anadditional three weeks. Embryos were assessed at each transfer for theformation of callus resembling embryogenic callus characterized byclear, glassy, projections consisting of elongated cells subtended withdense head cells resembling a somatic embryo. To date, up to 4% of theembryos bombarded with pUC4CLGUS and pBI4CLAX have formed embryogeniccallus on kanamycin containing medium.

[0091] Histochemical screening with x-gluc to detect GUS activity ofembryogenic calli derived from embryos bombarded with the pUC4CLGUSconstruct has identified 22 interior spruce and one Sitka sprucetransformed embryogenic lines. The GUS staining of these lines issurprisingly strong. Over 20 embryogenic lines derived from embryosbombarded with pBI4CLAX grew on kanamycin containing medium.

References Cited

[0092] 1. Goldschmidt, E. E. and S. C. Huber. 1992. Regulation ofPhotosynthesis by End-Product Accumulation in Leaves of Plants StoringStarch, Sucrose and Hexose Sugars. Plant Physiol. 99:1443-1448.

[0093] 2. Sonnewald, U. and L. Willmitzer. 1992. Molecular approaches tosink-source interactions. Plant Physiol. 99:1267-1270.

[0094] 3. Shewmaker, C. K. and D. M. Stalker. 1992. Modifying starchbiosynthesis with transgenes in potatoes. Plant Physiol. 100:1083-1086.

[0095] 4. Delmer, D. P. 1987. Cellulose biosynthesis. Ann. Rev. PlantPhysiol. 38:259-290.

[0096] 5. Delmer, D. P. and Y. Amor. 1995. Cellulose Biosynthesis. PlantCell 7:987-1000.

[0097] 6. Ross, P., R. Mayer, M. Benziman. 1991. Cellulose biosynthesisand function in bacteria. Microbiol. Rev. 55:35-58.

[0098] 7. Betlach M. R., D. H. Doherty, R. W. Vanderslice. 1987. Processfor the synthesis of sugar nucleotides using recombinant DNA methods.International Patent WO 87/05937.

[0099] 8. Farnum, P., R. Timmis, J. K. Kulp. 1983. Biotechnology offorest yield. Science 219:694-702.

[0100] 9. Sambrook J., E. F. Fritsch and T. Maniatis. 1989. MolecularCloning. 2nd Ed. Cold Spring Harbour Laboratory Press: 18. pp. 3-86.

1 11 1 46 DNA Artificial Sequence Description of Artificial Sequence5′-Primer 1 taggatccgt cgacc atg gtc aag ccc ctt aaa aaa gcc gta ttg c 46Met Val Lys Pro Leu Lys Lys Ala Val Leu 1 5 10 2 10 PRT ArtificialSequence Description of Artificial Sequence5′- Primer 2 Met Val Lys ProLeu Lys Lys Ala Val Leu 1 5 10 3 52 DNA Acetobacter xylinum CDS(16)..(48) 3 ttgaggtaaa tatta gtg att aag ccc ctt aaa aaa gcc gta ttgccg gttg 52 Val Ile Lys Pro Leu Lys Lys Ala Val Leu Pro 1 5 10 4 11 PRTAcetobacter xylinum 4 Val Ile Lys Pro Leu Lys Lys Ala Val Leu Pro 1 5 105 48 DNA Acetobacter xylinum 5 ggtgccggaa gatcacttgt acttcgtcaggaattcacgc acgccggg 48 6 6 PRT Acetobacter xylinum 6 Ser Asn Val Cys AlaPro 1 5 7 48 DNA Artificial Sequence Description of ArtificialSequence3′-Primer 7 ggtgcctcta gatcacttgt acttcgtcag gacttcacgc acgccggg48 8 334 DNA Acetobacter xylinum CDS (25)..(333) 8 tcatgcatct tgaggtaaatatta gtg att aag ccc ctt aaa aaa gcc gta 51 Val Ile Lys Pro Leu Lys LysAla Val 1 5 ttg ccg gtt gcc ggc ctt gga aca cgc ttt ctg ccc gcc acc aagtgc 99 Leu Pro Val Ala Gly Leu Gly Thr Arg Phe Leu Pro Ala Thr Lys Cys10 15 20 25 gtg ccc aag gaa atg ctg acc gtt gtt gac cgt ccg ctg atc cagtat 147 Val Pro Lys Glu Met Leu Thr Val Val Asp Arg Pro Leu Ile Gln Tyr30 35 40 gcg att gac gag gca cgc gaa gcc ggg atc gag gaa ttc tgc ctc gtt195 Ala Ile Asp Glu Ala Arg Glu Ala Gly Ile Glu Glu Phe Cys Leu Val 4550 55 tcc agc cgg ggc aag gat tcc ctg atc gat tat ttc gac att tcc tac243 Ser Ser Arg Gly Lys Asp Ser Leu Ile Asp Tyr Phe Asp Ile Ser Tyr 6065 70 gaa ctc gaa gac acg ctg aag gcc cgc aag aag aca tcg gca ctg aag291 Glu Leu Glu Asp Thr Leu Lys Ala Arg Lys Lys Thr Ser Ala Leu Lys 7580 85 gcc ctg gaa gca acc cgc gtc atc ccg ggc acc atg ctg tcc g 334 AlaLeu Glu Ala Thr Arg Val Ile Pro Gly Thr Met Leu Ser 90 95 100 9 103 PRTAcetobacter xylinum 9 Val Ile Lys Pro Leu Lys Lys Ala Val Leu Pro ValAla Gly Leu Gly 1 5 10 15 Thr Arg Phe Leu Pro Ala Thr Lys Cys Val ProLys Glu Met Leu Thr 20 25 30 Val Val Asp Arg Pro Leu Ile Gln Tyr Ala IleAsp Glu Ala Arg Glu 35 40 45 Ala Gly Ile Glu Glu Phe Cys Leu Val Ser SerArg Gly Lys Asp Ser 50 55 60 Leu Ile Asp Tyr Phe Asp Ile Ser Tyr Glu LeuGlu Asp Thr Leu Lys 65 70 75 80 Ala Arg Lys Lys Thr Ser Ala Leu Lys AlaLeu Glu Ala Thr Arg Val 85 90 95 Ile Pro Gly Thr Met Leu Ser 100 10 317DNA Acetobacter xylinum CDS (8)..(316) 10 cggtacc atg gtc aag ccc cttaaa aaa gcc gta ttg ccg gtt gcc ggc 49 Met Val Lys Pro Leu Lys Lys AlaVal Leu Pro Val Ala Gly 1 5 10 ctt gga aca cgc ttt ctg ccc gcc acc aagtgc gtg ccc aag gaa atg 97 Leu Gly Thr Arg Phe Leu Pro Ala Thr Lys CysVal Pro Lys Glu Met 15 20 25 30 ctg acc gtt gtt gac cgt ccg ctg atc cagtat gcg att gac gag gca 145 Leu Thr Val Val Asp Arg Pro Leu Ile Gln TyrAla Ile Asp Glu Ala 35 40 45 cgc gaa gcc ggg gtc gag gaa ttc tgc ctc gtttcc agc cgg ggc aag 193 Arg Glu Ala Gly Val Glu Glu Phe Cys Leu Val SerSer Arg Gly Lys 50 55 60 gat tcc ctg atc gat tat ttc gac att tca tac gaactc gaa gac acg 241 Asp Ser Leu Ile Asp Tyr Phe Asp Ile Ser Tyr Glu LeuGlu Asp Thr 65 70 75 ctg aag gcc cgc aag aag aca tcg gca ctg aag gcc ctggaa gca acc 289 Leu Lys Ala Arg Lys Lys Thr Ser Ala Leu Lys Ala Leu GluAla Thr 80 85 90 cgc gtc atc ccg ggc acc atg ttg tcc g 317 Arg Val IlePro Gly Thr Met Leu Ser 95 100 11 103 PRT Acetobacter xylinum 11 Met ValLys Pro Leu Lys Lys Ala Val Leu Pro Val Ala Gly Leu Gly 1 5 10 15 ThrArg Phe Leu Pro Ala Thr Lys Cys Val Pro Lys Glu Met Leu Thr 20 25 30 ValVal Asp Arg Pro Leu Ile Gln Tyr Ala Ile Asp Glu Ala Arg Glu 35 40 45 AlaGly Val Glu Glu Phe Cys Leu Val Ser Ser Arg Gly Lys Asp Ser 50 55 60 LeuIle Asp Tyr Phe Asp Ile Ser Tyr Glu Leu Glu Asp Thr Leu Lys 65 70 75 80Ala Arg Lys Lys Thr Ser Ala Leu Lys Ala Leu Glu Ala Thr Arg Val 85 90 95Ile Pro Gly Thr Met Leu Ser 100

1. A process of increasing plant growth and yield which comprisesintroducing into a plant a DNA sequence encoding a product whichmodifies, in the plant, the level of cellulose precursors.
 2. Theprocess of claim 1 wherein the product is selected from the groupconsisting of enzymes, proteins and ribonucleic acid.
 3. The process ofclaim 1 wherein the product is a carbohydrate modifying enzyme selectedfrom the group consisting of uridine diphosphate glucosepyrophosphorylase (UDPG-PPase), sucrose synthetase and cellulosesynthetase.
 4. The process of claim 1 wherein the product regulates theexpression of a cellulose-dependent enzyme.
 5. The process of claim 1wherein the precursor is uridine diphosphate glucose (UDP-glucose). 6.The process of claim 1 wherein the plant is a tree.
 7. The process ofclaim 1 wherein the DNA sequence comprises the UDPG-PPase gene derivedfrom a bacterium.
 8. The process of claim 1 wherein the DNA sequencecomprises the UDPG-PPase gene derived from Acetobacter xylinum.
 9. Aprocess of modifying the production of cellulose in a plant whichcomprises introducing into and expressing in said plant a DNA sequenceencoding a product which modifies the level of cellulose precursors inthe plant.
 10. The process of claim 9 wherein the product is selectedfrom the group consisting of enzymes, proteins and ribonucleic acid. 11.The process of claim 9 wherein the product is a carbohydrate modifyingenzyme selected from the group consisting of UDPG-PPase, sucrosesynthetase and cellulose synthetase.
 12. The process of claim 9 whereinthe precursor is UDP-glucose.
 13. The process of claim 9 wherein theplant is a tree.
 14. A process of modifying the production of cellulosein a plant which comprises introducing into and expressing in said planta DNA sequence comprising the UDPG-PPase gene operably linked to anexpression effecting DNA sequence and flanked by translational start andstop signals.
 15. The process of claim 14 wherein the expressioneffecting DNA sequence is a promoter directing expression of theUDPG-PPase gene primarily in cellulose-producing cells of the plant. 16.The process of claim 14 wherein the expression effecting DNA sequence isa promoter selected from the group consisting of one or more of CaMV 35Sand 4CL from parsley.
 17. The process of claim 14 wherein the plant is atree.
 18. A plant having modified cellulose producing activity as aresult of introducing into said plant or parent of said plant a DNAsequence coding for a product which modifies the level of celluloseprecursors in the plant.
 19. The plant of claim 18 wherein the productis an enzyme.
 20. The plant of claim 18 wherein the product is acarbohydrate modifying enzyme selected from the group consisting ofUDPG-PPase, sucrose synthetase and cellulose synthetase.
 21. The plantof claim 18 wherein the product regulates the expression of acellulose-dependent enzyme.
 22. The plant of claim 18 wherein theprecursor is UDP-glucose.
 23. The plant of claim 18 being a tree. 24.The plant of claim 18 wherein the DNA sequence comprises the UDPG-PPasegene derived from a bacterium.
 25. A DNA expression vector comprising aDNA sequence encoding a bacterial protein which modifies the level ofcellulose precursors, said DNA sequence being operably linked to anexpression effecting DNA sequence and flanked by translational start andstop sequences.
 26. A genetically modified seed product comprising a DNAsequence encoding a product capable of modifying the level of celluloseprecursors in the plant resulting from the seed product.